Inhibitors of cognitive decline

ABSTRACT

Compounds that are central nervous system drug candidates for the treatment of cognitive decline and, more particularly, Alzheimer&#39;s disease are provided. Methods of treating, inhibiting, and/or abatement of cognitive decline and/or Alzheimer&#39;s disease with a compound or pharmaceutically acceptable salt of the invention are also provided. Also provided are methods of preparing the compounds/compositions of the invention.

This application is a continuation of U.S. patent application Ser. No.13/388,128, with a 371 (c) date of Jan. 31, 2012, which is a U.S.national stage filing under 35 U.S.C. §371 of International ApplicationNo. PCT/US2010/044136 filed on Aug. 2, 2010, which claims the benefit ofand priority to U.S. provisional patent application Ser. No. 61/230,326filed on Jul. 31, 2009, and to U.S. provisional patent application Ser.No. 61/308,686 filed on Feb. 26, 2010, each of which is herebyincorporated by reference in its entirety.

This invention was made with support from the U.S. government under agrant from the U.S. National Institute of Health, gran number1R43AG037337-01. The U.S. government has certain rights in thisinvention.

SUMMARY

The present invention provides, inter alia, compounds and methods forpreparation thereof useful for inhibiting, treating, or abatement ofcognitive decline. In a method called “chemical conditioning”, certaincompounds of the present invention are derived from naturally occurringcompounds, such as those found in turmeric (Curcuma longa). The chemicalconditioning process described herein is applicable to a large varietyof biological extracts and may be used to create compound arrays forscreening for potential new drug candidates. Further, in general,compounds derived by the chemical conditioning process are chemicallystable and structurally diverse, and good candidates for use in drugscreenings for pharmaceutical activity. In some embodiments, compoundsderived from turmeric oil are provided. According to some embodiments ofthe invention, compounds derived from turmeric oil by the chemicalconditioning process described herein are provided. In anotherembodiment, the invention provides a method of preparing an array ofchemical compounds from turmeric oil. In some other embodiments,compounds useful for inhibiting, treating, or abatement of cognitivedecline are prepared by chemical synthesis.

In some embodiments, the present invention provides compounds of FormulaI-0, I, Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1, Ia-2-0, Ia-2, Ib-1-0, Ib-1,Ib-2-0, or Ib-2:

or pharmaceutically acceptable salts, wherein constituent members areprovided below.

The present invention further provides pharmaceutical compositionscomprising a compound of present invention (such as a derivative ofturmeric oil or a compound of Formula I-0, I, Ia-0, Ia, Ib-0, Ib,Ia-1-0, Ia-1, Ia-2-0, Ia-2, Ib-1-0, Ib-1, Ib-2-0, or Ib-2), orpharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier. In some embodiments, the presentinvention further provides pharmaceutical compositions comprising acompound of Formula I-0, I, Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1, Ia-2-0,Ia-2, Ib-1-0, Ib-1, Ib-2-0, or Ib-2, or pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable carrier.

The present invention further provides methods of inhibiting, treating,and/or abating cognitive decline and/or Alzheimer's disease with acompound of present invention such as a compound of Formula I-0, I,Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1, Ia-2-0, Ia-2, Ib-1-0, Ib-1, Ib-2-0, orIb-2, or pharmaceutically acceptable salt of the same.

The present invention further provides methods of inhibiting, treating,or abatement of cognitive decline with a compound of present inventionsuch as a compound of Formula I-0, I, Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1,Ia-2-0, Ia-2, Ib-1-0, Ib-1, Ib-2-0, or Ib-2, or pharmaceuticallyacceptable salt of the same.

The present invention further provides methods of inhibiting, treating,or abatement of one or more of amyloid production, amyloid assembly,amyloid aggregation, amyloid binding (to cells in the brain such asneuron cells), the activity/effect of Abeta oligomers on neurons, andamyloid deposition (on cells in the brain such as neuron cells) with acompound of present invention such as a compound of Formula I-0, I,Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1, Ia-2-0, Ia-2, Ib-1-0, Ib-1, Ib-2-0, orIb-2, or pharmaceutically acceptable salt of the same.

The present invention further provides compounds of Formula I-0, I,Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1, Ia-2-0, Ia-2, Ib-1-0, Ib-1, Ib-2-0, orIb-2, or pharmaceutically acceptable salts thereof, for use in therapy.

The present invention further provides use of the compounds of FormulaI-0, I, Ia-0, Ia, Ib-0, Ib, Ia-1-0, Ia-1, Ia-2-0, Ia-2, Ib-1-0, Ib-1,Ib-2-0, or Ib-2, or pharmaceutically acceptable salts thereof, for themanufacture/preparation of a medicament for use in therapy.

In some embodiments, the compounds of present invention inhibit, treat,or abate (partially inhibit) binding of the amyloid (including Abetaoligomers) to neurons (such as neurons in the brain) and are useful forthe inhibition, treatment, and abatement of cognitive decline and/orAlzheimer's disease. In some embodiments, the compounds of presentinvention inhibit, treat, or abate one or more of amyloid aggregation,amyloid binding, and amyloid deposition. In some embodiments, thecompounds of present invention inhibit, treat, or abate amyloidaggregation. In some embodiments, the compounds of present inventioninhibit, treat, or abate amyloid binding. In some embodiments, thecompounds of present invention inhibit, treat, or abate amyloiddeposition. In some embodiments, the compounds of present inventioninhibit, treat, or abate the activity/effect of Abeta oligomers onneurons. In some embodiments, the compounds show activity in abeta-secretase assay and are potentially useful for the inhibition,treatment, and abatement of cognitive decline and Alzheimer's disease.In some embodiments the derivative of turmeric oil is a compound inpurified and isolated form (for example, with a purity of greater than80%, 85%, 90%, 95%, 98%, or 99% by weight). The compounds and methodsdescribed herein may be used to treat one or more symptoms of cognitivedecline and/or Alzheimer's disease such as memory loss, confusion,impaired judgment, personality changes, disorientation, and loss oflanguage skills. Further, the compounds and methods described herein maybe useful in inhibiting, treating, and/or abating cognitive declineand/or Alzheimer's disease by restoring long term potentiation, and/orinhibiting, treating, or abatement of one or both of neurodegenerationand general amyloidosis, more specifically, by inhibiting, treating, orabatement of one or more of amyloid production, amyloid assembly,amyloid aggregation, amyloid binding, and amyloid deposition.

DESCRIPTION OF DRAWINGS

FIG. 1 shows results of an MTT assay in the presence and absence of aprocessed product of amyloid precursor protein.

FIG. 2 shows inhibition of processed product of amyloid precursorprotein-mediated membrane trafficking effect by CT0109.

FIG. 3 shows CT0109 inhibiting the memory loss effects of a processedproduct of amyloid precursor protein.

DETAILED DESCRIPTION

Cognitive decline, such as memory loss, confusion, impaired judgment,personality changes, disorientation, and loss of language skills occursin much of the population as they age, in varying degree. The mostcommon, severe and irreversible form of cognitive decline is Alzheimer'sdisease, which, at present, is always fatal.

The symptoms of cognitive decline and Alzheimer's disease are thought tostem from the formation of amyloid plaques and neurofibrillary tangles,which are thought to contribute to the degradation of the neurons (nervecells) in the brain and the subsequent onset of symptoms. Amyloid is ageneral term for protein fragments that the body produces normally.Beta-amyloid is a fragment of a protein that is snipped from anotherprotein called amyloid precursor protein (APP). In a healthy brain,beta-amyloid protein fragments are broken down and eliminated. Inindividuals with Alzheimer's disease and other forms of cognitivedecline, the fragments accumulate to form hard, insoluble plaques.Neurofibrillary tangles are insoluble twisted fibers that are foundinside of the brain's cells. The protein contained in neurofibrillarytangles, i.e., the tau protein, forms a microtubule, which helpstransport nutrients and other important substances from one part of thenerve cell to another. In Alzheimer's disease the tau protein isabnormal and the microtubule structures collapse.

Beta-secretase is the enzyme in the human brain responsible for theproduction of Beta-amyloid, the pathogenic substance responsible for theformation of brain plaques and tangles in the Alzheimer's diseasedbrain. Beta-amyloid and its oligomers (beta-amyloid oligomers or Abetaoligomers) are also believed to be responsible for early cognitivedecline in the pre-Alzheimer's diseased brain. Inhibition ofbeta-secretase would be expected to lessen beta-amyloid burden in thebrain and thus slow cognitive decline, block the formation of amyloidoligomers, the production of plaques and tangles, haltneurodegeneration, and to potentially treat mild cognitive impairmentand more serious forms of cognitive impairment such as Alzheimer'sdisease.

Millions of people worldwide are affected by cognitive decline andAlzheimer's disease. Accordingly, there is strong need to discoverinhibitors of cognitive decline, and in particular, compounds that areuseful in the treatment and abatement of cognitive decline andAlzheimer's disease, by methods such as inhibiting amyloid (includingAbeta oligomers) production, amyloid (including Abeta oligomers)aggregation, and/or amyloid (including Abeta oligomers) deposition(i.e., plaqing), inhibiting neuorodegeneration, and/or restoring longterm potentiation, and/or inhibiting the activity/effect of Abetaoligomers on neurons. There is also a need for inhibitors of cognitivedecline that are chemically and biologically stable.

Plants have attracted relatively little attention as potentiallyvaluable resources for drug discovery in the area of cognitive declineand Alzheimer's disease. The use of plant extracts to produce unnaturalderivatives of compounds of medicinal interest is not generally used.Accordingly, there is also a need for a method of producing compounds ofmedicinal interest from plant extracts and extracts from otherbiological sources. In particular, there is also a need to produce andidentify compounds derived from plant extracts that are useful in thetreatment and abatement of cognitive decline and Alzheimer's disease.

Turmeric—a highly reputed herb in Indian system of medicine-Ayurveda—isthe rhizome of Curcuma longa L. Syn. Curcuma domestica Valeton (Fam.Zingiberaceae), which grows abundantly in India. It has long been usedas a spice and a coloring agent in food. Its powder or extracts arerecommended to treat wounds and inflammation. Lipid soluble extracts ofrhizomes and leaves of Curcuma species of Zingiberaceae family arereported to be useful for the treatment of neurocerebrovasculardisorders. See WO 2003051380. Turmeric oil can be extracted fromturmeric (Curcuma longa) with supercritical carbon dioxide. See e.g. B.Gopalan, et. al, “Supercritical Carbon Dioxide Extraction of Turmeric(Curcuma longa)”, J. Agric. Food Chem., 2000, 48 (6), pp 2189-2192; seealso L-H Chang, et. al, “Supercritical carbon dioxide extraction ofturmeric oil from Curcuma longa Linn and purification of turmerones”,Separation and Purification Technology Volume 47, Issue 3, January 2006,Pages 119-125. The present invention, in part, relates to producing andidentifying compounds derived from Turmeric oil (i.e. Turmeric oilderivatives) that are useful in the treatment and abatement of cognitivedecline and Alzheimer's disease. The present invention also, in part,relates to chemically synthesizing compounds that are useful in thetreatment and abatement of cognitive decline and Alzheimer's disease.

The compounds, compositions, and methods described herein are directedtoward these needs and other ends.

Embodiments of the present invention provide, inter alia, a compound ofFormula I-0:

or pharmaceutically acceptable salt thereof, wherein:

is a single bond or a double bond;

R1 is H, CH3, CF3, F, Cl, Br, or —OCF3;

R2 is H, C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl, or C6-10 aryl,wherein each of the C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl, orC6-10 aryl is substituted by 0, 1, 2, 3, 4, or 5 substituents eachindependently selected from OH, amino, halo, C1-6 alkyl, C1-6 haloalkyl,C1-6 alkoxy, and C1-6 haloalkoxy;

R3 is OH or NR3aNR3b;

R3a is H, C1-6 alkyl, C1-6 haloalkyl, cycloalkylalkyl, C3-7 cycloalkyl,arylalkyl, or C6-10 aryl, wherein each of the C1-6 alkyl, C1-6haloalkyl, C3-7 cycloalkyl, arylalkyl, or C6-10 aryl is substituted by0, 1, 2, 3, 4, or 5 substituents each independently selected from OH,amino, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, and C1-6haloalkoxy;

R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl, C₃₋₇cycloalkyl, arylalkyl, or C₆₋₁₀ aryl, wherein each of the C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl is substituted by 0, 1,2, 3, 4, or 5 substituents each independently selected from OH, amino,halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy;

or R^(3a) and R^(3b) together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group that issubstituted with 0, 1, 2, 3, 4, or 5 substituents each independentlyselected from OH, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, aryl, arylalkyl, heteroaryl, heteroarylalkyl,cycloalkyl, and heterocycloalkyl; and

R⁴ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl,wherein each of the C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, orC₆₋₁₀ aryl is substituted by 0, 1, 2, 3, 4, or 5 substituents eachindependently selected from OH, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.

In some embodiments, when

is a double bond and R³ is OH, then at least one of R′, R², and R⁴ isother than H. In some embodiments, the compound of Formula I-0 is otherthan 2-methyl-6-p-tolylhept-2-en-4-ol.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula I:

or pharmaceutically acceptable salt thereof

In some embodiments, the compound of Formula I is other than(6S)-2-methyl-6-p-tolylhept-2-en-4-ol.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ia-0:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ia:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ib-0:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ib:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of FormulaIa-1-0:

or pharmaceutically acceptable salt thereof

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ia-1:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of FormulaIa-2-0:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ia-2:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of FormulaIb-1-0:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ib-1:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of FormulaIb-2-0:

or pharmaceutically acceptable salt thereof.

In some embodiments, the compound of the present invention orpharmaceutically acceptable salt thereof is a compound of Formula Ib-2:

or pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is H, CH₃, or CF₃. In some further embodiments,R¹ is CH₃ or CF₃. In yet further embodiments, R¹ is CH₃.

In some embodiments, R¹ is H or CH₃.

In some embodiments, R¹ is F, Cl, or Br. In other embodiments, R¹ isOCF₃.

In some embodiments, R² is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl. In some further embodiments, R² is H, C₁₋₆alkyl, or C₃₋₇ cycloalkyl.

In some embodiments, R² is H or C₁₋₆ alkyl. In some further embodiments,R² is H or methyl. In yet further embodiments, R² is H.

In some embodiments, R¹ is H, CH₃, or CF₃; and R² is H or C₁₋₆ alkyl. Insome further embodiments, R¹ is CH₃ or CF₃; and R² is H. In yet furtherembodiments, R¹ is CH₃; and R² is H.

In some embodiments, R^(3a) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,cycloalkylalkyl, C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl, wherein eachof the C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl, C₃₋₇ cycloalkyl,arylalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3, 4, or 5substituents each independently selected from halo, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy. In some furtherembodiments, R^(3a) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl,C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl.

In some embodiments, R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,cycloalkylalkyl, C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl, wherein eachof the C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl, C₃₋₇ cycloalkyl,arylalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3, 4, or 5substituents each independently selected from halo, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy. In some furtherembodiments, R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl,C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl.

In some embodiments, R^(3a) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl, wherein each of the C₁₋₆ alkyl, C₁₋₆haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3,4, or 5 substituents each independently selected from OH, amino, halo,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.

In some embodiments, R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl, wherein each of the C₁₋₆ alkyl, C₁₋₆haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3,4, or 5 substituents each independently selected from OH, amino, halo,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.

In some embodiments, R^(3a) is H; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, cycloalkylalkyl, C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl,wherein each of the C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl, C₃₋₇cycloalkyl, arylalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3, 4, or5 substituents each independently selected from OH, amino, halo, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy. In some furtherembodiments, R^(3a) is H; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,cycloalkylalkyl, C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl, wherein eachof the C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkylalkyl, C₃₋₇ cycloalkyl,arylalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3, 4, or 5substituents each independently selected from halo, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.

In some embodiments, R^(3a) is H; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, cycloalkylalkyl, C₃₋₇ cycloalkyl, arylalkyl, or C₆₋₁₀ aryl.

In some embodiments, R^(3a) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl.

In some embodiments, R^(3a) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl;

In some embodiments, R^(3a) is H; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, or C₃₋₇ cycloalkyl.

In some embodiments, R^(3a) is H; and R^(3b) is C₁₋₆ alkyl or C₁₋₆haloalkyl.

In some embodiments, R^(3a) is H; and R^(3b) is C₁₋₆ alkyl.

In some embodiments, R^(3a) and R^(3b) together with the N atom to whichthey are attached form pyrrolidinyl, piperidinyl, piperazinyl, ormorpholinyl, each substituted with 0, 1, 2, 3, 4, or 5 substituents eachindependently selected from OH, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, aryl, arylalkyl, heteroaryl,heteroarylalkyl, cycloalkyl, and heterocycloalkyl.

In some embodiments, R^(3a) and R^(3b) together with the N atom to whichthey are attached form pyrrolidinyl, piperidinyl, piperazinyl, ormorpholinyl, each substituted with 0, 1, 2, 3, 4, or 5 substituents eachindependently selected from OH, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, phenyl, and benzyl.

In some embodiments, R⁴ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl. In some further embodiments, R⁴ is H, C₁₋₆alkyl, or C₃₋₇ cycloalkyl.

In some embodiments, R⁴ is H or C₁₋₆ alkyl. In some further embodiments,R⁴ is H or methyl. In yet further embodiments, R⁴ is H.

In some embodiments,

In some embodiments, R² is H or C₁₋₆ alkyl, and R⁴ is H or C₁₋₆ alkyl.

In some embodiments, R² is H or methyl, and R⁴ is H or methyl.

In some embodiments, R¹ is H, CH₃, or CF₃; R² is H or C₁₋₆ alkyl, and R⁴is H or C₁₋₆ alkyl. In some further embodiments, R¹ is CH₃ or CF₃; R² isH; and R⁴ is H. In yet further embodiments, R¹ is CH₃; R² is H; and R⁴is H.

At various places in the present specification, substituents ofcompounds of the invention are disclosed in groups or in ranges. It isspecifically intended that embodiments the invention include each andevery individual subcombination of the members of such groups andranges. For example, the term “C₁₋₆ alkyl” is specifically intended toindividually disclose methyl (C₁ alkyl), ethyl (C₂ alkyl), C₃ alkyl, C₄alkyl, C₅ alkyl, and C₆ alkyl.

For compounds of the invention in which a variable appears more thanonce, each variable can be a different moiety selected from the Markushgroup defining the variable. For example, where a structure is describedhaving two R groups that are simultaneously present on the samecompound, then the two R groups can represent different moietiesselected from the Markush group defined for R.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

The term “n-membered” where n is an integer typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, pyridine is an example of a6-membered heteroaryl ring and thiophene is an example of a 5-memberedheteroaryl group.

As used herein, the term “alkyl” is meant to refer to a saturatedhydrocarbon group which is straight-chained or branched. Example alkylgroups include, but are not limited to, methyl (Me), ethyl (Et), propyl(e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl,t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.An alkyl group can contain from 1 to about 20, from 2 to about 20, from1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4,or from 1 to about 3 carbon atoms.

As used herein, “haloalkyl” refers to an alkyl group having one or morehalogen substituents. Example haloalkyl groups include, but are notlimited to, CF₃, C₂F₅, CHF₂, CCl₃, CHCl₂, C₂Cl₅, CH₂CF₃, and the like.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and thelike. In some embodiments, aryl groups have from 6 to about 20 carbonatoms. In some embodiments, aryl groups have from 6 to about 10 carbonatoms.

As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbonsincluding cyclized alkyl, alkenyl, and alkynyl groups that contain up to20 ring-forming carbon atoms. Cycloalkyl groups can include mono- orpolycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well asspiro ring systems. A cycloalkyl group can contain from 3 to about 15,from 3 to about 10, from 3 to about 8, from 3 to about 6, from 4 toabout 6, from 3 to about 5, or from 5 to about 6 ring-forming carbonatoms. Ring-forming carbon atoms of a cycloalkyl group can be optionallysubstituted by oxo or sulfido. Example cycloalkyl groups include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl,cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and thelike. Also included in the definition of cycloalkyl are moieties thathave one or more aromatic rings fused (i.e., having a bond in commonwith) to the cycloalkyl ring, for example, benzo or thienyl derivativesof pentane, pentene, hexane, and the like (e.g.,2,3-dihydro-1H-indene-1-yl, or 1H-inden-2(3H)-one-1-yl). Preferably,“cycloalkyl” refers to cyclized alkyl groups that contain up to 20ring-forming carbon atoms. Examples of cycloalkyl preferably includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,adamantyl, and the like.

As used herein, “heteroaryl” groups refer to an aromatic heterocyclehaving up to 20 ring-forming atoms and having at least one heteroatomring member (ring-forming atom) such as sulfur, oxygen, or nitrogen. Insome embodiments, the heteroaryl group has at least one or moreheteroatom ring-forming atoms each independently selected from sulfur,oxygen, and nitrogen. Heteroaryl groups include monocyclic andpolycyclic (e.g., having 2, 3 or 4 fused rings) systems. Examples ofheteroaryl groups include without limitation, pyridyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl,thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl,benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl,tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl,purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In someembodiments, the heteroaryl group has from 1 to about 20 carbon atoms,and in further embodiments from about 1 to about 5, from about 1 toabout 4, from about 1 to about 3, from about 1 to about 2, carbon atomsas ring-forming atoms. In some embodiments, the heteroaryl groupcontains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. Insome embodiments, the heteroaryl group has 1 to about 4, 1 to about 3,or 1 to 2 heteroatoms.

As used herein, “heterocycloalkyl” refers to non-aromatic heterocycleshaving up to 20 ring-forming atoms including cyclized alkyl, alkenyl,and alkynyl groups where one or more of the ring-forming carbon atoms isreplaced by a heteroatom such as an O, N, or S atom. Hetercycloalkylgroups can be mono or polycyclic (e.g., both fused and spiro systems).Example “heterocycloalkyl” groups include morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, pyrrolidin-2-one-3-yl, andthe like. Ring-forming carbon atoms and heteroatoms of aheterocycloalkyl group can be optionally substituted by oxo or sulfido.For example, a ring-forming S atom can be substituted by 1 or 2 oxo[i.e., form a S(O) or S(O)₂]. For another example, a ring-forming C atomcan be substituted by oxo (i.e., form carbonyl). Also included in thedefinition of heterocycloalkyl are moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thenonaromatic heterocyclic ring, for example pyridinyl, thiophenyl,phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles suchas indolene, isoindolene, isoindolin-1-one-3-yl,4,5,6,7-tetrahydrothieno[2,3-c]pyridine-5-yl,5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one-5-yl, and3,4-dihydroisoquinolin-1(2H)-one-3yl groups. Ring-forming carbon atomsand heteroatoms of the heterocycloalkyl group can be optionallysubstituted by oxo or sulfido. In some embodiments, the heterocycloalkylgroup has from 1 to about 20 carbon atoms, and in further embodimentsfrom about 3 to about 20 carbon atoms. In some embodiments, theheterocycloalkyl group contains 3 to about 14, 3 to about 7, or 5 to 6ring-forming atoms. In some embodiments, the heterocycloalkyl group has1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments,the heterocycloalkyl group contains 0 to 3 double bonds. In someembodiments, the heterocycloalkyl group contains 0 to 2 triple bonds.

As used herein, “halo” or “halogen” includes fluoro, chloro, bromo, andiodo.

As used herein, “alkoxy” refers to an —O-alkyl group. Example alkoxygroups include methoxy, ethoxy, propoxy (e.g., n-propoxy andisopropoxy), t-butoxy, and the like.

As used herein, “haloalkoxy” refers to an —O-haloalkyl group. An examplehaloalkoxy group is OCF₃. As used herein, “trihalomethoxy” refers to amethoxy group having three halogen substituents. Examples oftrihalomethoxy groups include, but are not limited to, —OCF₃, —OCClF₂,—OCCl₃, and the like.

As used herein, “arylalkyl” refers to a C₁₋₆ alkyl substituted by aryl.Example arylalkyl groups include, but are not limited to, C₁₋₆ alkylsubstituted by C₆₋₁₀ aryl (e.g. benzyl).

As used herein, “cycloalkylalkyl” refers to C₁₋₆ alkyl substituted bycycloalkyl. Example cycloalkylalkyl groups include, but are not limitedto, C₁₋₆ alkyl substituted by C₃₋₁₀ cycloalkyl or C₃₋₇ cycloalkyl (e.g.cyclopropylmethyl).

As used herein, “amino” refers to NH₂.

As used herein, the term “optionally substituted” means thatsubstitution is optional and therefore includes both unsubstituted andsubstituted atoms and moieties. A “substituted” atom or moiety indicatesthat any hydrogen on the designated atom or moiety can be replaced witha selection from the indicated substituent group, provided that thenormal valency of the designated atom or moiety is not exceeded, andthat the substitution results in a stable compound. For example, if amethyl group (i.e., CH₃) is optionally substituted, then 3 hydrogenatoms on the carbon atom can be replaced with substituent groups.

The compounds described in the embodiments herein can be asymmetric(e.g., having one or more stereocenters). All stereoisomers, such asenantiomers and diastereomers, are intended unless otherwise indicated.Compounds of the present invention that contain asymmetricallysubstituted carbon atoms can be isolated in optically active or racemicforms. Methods on how to prepare optically active forms from opticallyactive starting materials are known in the art, such as by resolution ofracemic mixtures or by stereoselective synthesis. Many geometric isomersof olefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms. Where a compoundcapable of stereoisomerism or geometric isomerism is designated in itsstructure or name without reference to specific R/S or cis/transconfigurations, it is intended that all such isomers are contemplated.

Resolution of racemic mixtures (or mixture of diastereoisomers) ofcompounds can be carried out by any of numerous methods known in theart. An example method includes fractional recrystallizaion using achiral resolving acid which is an optically active, salt-forming organicacid. Suitable resolving agents for fractional recrystallization methodsare, for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids such as b-camphorsulfonic acid. Other resolvingagents suitable for fractional crystallization methods includestereoisomerically pure forms of α-methylbenzyl-amine (e.g., S and Rforms, or diastereomerically pure forms), 2-phenylglycinol,norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine,1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures (or mixture of diastereoisomers) can alsobe carried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Compounds of embodiments the invention also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond together with the concomitant migration of aproton. Tautomeric forms include prototropic tautomers which areisomeric protonation states having the same empirical formula and totalcharge. Example prototropic tautomers include ketone—enol pairs,amide—imidic acid pairs, lactam—lactim pairs, amide—imidic acid pairs,enamine—imine pairs, and annular forms where a proton can occupy two ormore positions of a heterocyclic system, for example, 1H- and3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium orsterically locked into one form by appropriate substitution.

Compounds of embodiments the invention further include hydrates andsolvates, as well as anhydrous and non-solvated forms.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric iosomers, tautomers, and isotopes of thestructures depicted.

All compounds and pharmaceutically acceptable salts thereof, can beprepared or present together with other substances such as water andsolvents (e.g. hydrates and solvates) or can be isolated.

Compounds of embodiments the invention can also include all isotopes ofatoms occurring in the intermediates or final compounds. Isotopesinclude those atoms having the same atomic number but different massnumbers. For example, isotopes of hydrogen include tritium anddeuterium.

In some embodiments, the compounds of the invention, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which is formed or detected. Partial separation caninclude, for example, a composition enriched in the compound of theinvention. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compound of the invention, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

Compounds of embodiments the invention are intended to include compoundswith stable structures. As used herein, “stable compound” and “stablestructure” are meant to indicate a compound that is sufficiently robustto survive isolation to a useful degree of purity from a reactionmixture, and formulation into an efficacious therapeutic agent.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions, “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g. a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, for example, a temperature fromabout 20° C. to about 30° C.

Embodiments of the present invention also includes pharmaceuticallyacceptable salts of the compounds described herein. As used herein,“pharmaceutically acceptable salts” refers to derivatives of thedisclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts of the present inventioninclude the conventional non-toxic salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile(ACN) are preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety.

Chemical Conditioning

In some embodiments, a method of preparing an array of chemicalcompounds from a biological extract such as Turmeric oil is provided.

The method of the invention, termed “chemical conditioning” is generallyapplicable to all biological extracts, in particular, natural plantextracts, common or medicinal. See e.g. US20080193574 and WO2008042755,each of which is incorporated herein by reference in its entirety.Chemical conditioning is a method which produces novel unnaturaldrug-like compounds from readily available natural materials. Ingeneral, the “chemical conditioning” of natural extracts coupled withpre-fractionation of the chemically conditioned extracts facilitatessuccessful biochemical screening of extracts by destroying reactivenatural compounds that generate false positive results in biochemicalassays. Chemical conditioning produces novel lead-like and drug-likecompounds and, the reductive amination protocol and reduction protocoldescribed here can produce structurally diverse nitrogen-containingproducts and alcohol products that are particularly lead-like anddrug-like.

In certain embodiments of the present invention, a method of preparingchemical compounds from a biological extract is exemplified in Scheme 1below. According to the method, first, a biological extract, e.g., aplant extract is provided, the biological extract has one or morebiological compounds, each biological compound having one or morereactive electrophilic groups. Next, the biological compounds in thebiological extract are reacted with an amine to incorporate the amineinto the biological compounds. Next, the biological compounds having theincorporated amine are reacted with a reducing agent to reduce theintermediate imine and enamine compounds and form one or morenitrogen-containing chemical compounds. Thus, the resultantnitrogen-containing chemical compounds are derivatives of the biologicalcompounds in the biological extract.

Similarly, in certain other embodiments of the present invention, amethod of preparing chemical compounds from a biological extract isexemplified in Scheme 1a below. A secondary amine (wherein each ofR^(1*) and R^(2*) can be alkyl or the like; or R^(1*) and R^(2*),together with the N atom to which they are attached, form a cyclic aminesuch as pyrollidine) is used in the conditioned extracts.

In some embodiments, the compounds in the conditioned extraction arereaction products of compounds having ketones and aldehydes with variousamines. This reaction is followed by reduction such as hydride reductionof the intermediate imines and enamines to provide secondary andtertiary amines. The reaction of ketones and aldehydes with amines,followed by reduction of the formed imines and enamines to provideamines, is known in the art.

In some embodiments, the compounds in the conditioned extraction arereaction products of compounds having ketones and aldehydes with areducing reagent such as a hydride reducing reagent (e.g. sodiumborohydride, lithium aluminum hydride, sodium triacetoxy borohydride).In some embodiments, the ketones and aldehydes are reduced to alcohols.In some embodiments, the compounds in the conditioned extract arecompounds having other reactive functional groups that can be reduced inthe presence of a reducing reagent such as a hydride reducing reagent(e.g. sodium borohydride, lithium aluminum hydride, sodium triacetoxyborohydride).

In some embodiments, the chemical conditioning method described hereinemploys a biological extract (such as Turmeric oil), using manydifferent reagents, to efficiently produce an array ofnitrogen-containing chemical compounds. The ready commercialavailability of many low molecular weight amines for use as inputs inthe reductive amination sequence enables the development of manydifferent and structurally diverse central nervous system druglikemixtures from the same natural extract. Suitable amines for use in thepresent method are selected from the group consisting of primary amines,secondary amines, cyclic amines, pyrollidine, and amino acids. Suitablereducing agents for use in the present method are selected from thegroup of hydride reducing agents including but not limited to sodiumborohydride, sodium triacetoxyborohydride, and lithium aluminum hydride.

In some embodiments, the chemical conditioning method described hereinemploys a biological extract (such as Turmeric oil), using one or morereducing reagents, to efficiently produce an array of alcohol-containingchemical compounds (alcohol derivatives). Suitable reducing agents foruse in the present method are selected from the group of hydridereducing agents including but not limited to sodium borohydride, sodiumtriacetoxyborohydride, and lithium aluminum hydride.

The method may further comprise quenching the reaction by using aquenching agent, wherein the quenching agent is selected from but notlimited to the group consisting of sodium bicarbonate, sodium carbonate,sodium sulfate, sodium sulfate decahydrate. The method may also furthercomprise isolating one or more of the nitrogen-containing chemicalcompounds, in a purified or unpurified form. The resultantnitrogen-containing chemical may then be screened or tested forbiological activity.

The process of chemical conditioning by reduction or reductive aminationdescribed herein destroys reactive electrophiles in the natural extract,including ketones, as in the Turmeric oils, and converts them tochemically stable compounds such as amines or alcohols. The resultingconditioned extracts contain both natural compounds and novel unnaturalnitrogen-containing amine products or alcohol products that arepotential drug candidates. In some embodiments, in the process ofchemical conditioning by reductive amination described herein, reactiveelectrophiles in the natural extract, including ketones, as in theTurmeric oils, are destroyed and the ketone compounds are converted toother chemical compounds such as amines. In some other embodiments, inthe process of chemical conditioning by reduction described herein,reactive electrophiles in the natural extract, including ketones, as inthe Turmeric oils, are destroyed and the ketone compounds are convertedto other chemical compounds such as alcohols.

In the case of the extracts of Turmeric oil, the nitrogen-containingamine derivatives and alcohol derivatives are potential central nervoussystem drugs.

For the purpose of this disclosure, the following terms have thefollowing meanings.

The term “biological compound” as used herein refers to a chemicalcompound that occurs in nature.

The term “biological extract” as used herein refers to an extract from abiological sample, such as a plant extract, or other extract fromorganic matter, containing chemical compounds that occur in nature.

The term “reactive electrophilic group” as used herein refers to an atomor group of atoms that has the ability to react with a nucleophile.

The term “nitrogen-containing derivative” as used herein representsthose derivatives containing a nitrogen atom, where the nitrogen atom isa substitution another atom, such as oxygen in the parent compound.

The term “alcohol derivative” as used herein represents thosederivatives containing a hydroxyl group, where the hydroxyl group isreduced from a carbonyl group in the parent compound (such as a ketoneor aldehyde parent compound).

In one embodiment, a specific example of the chemical conditioningprocess is shown in Scheme 2 below. Scheme 2 shows the two-stepreductive amination chemical conditioning protocol performed on Turmericoil in accordance with one embodiment of the method, wherein Turmericoil comprising ketones 2-1a and 2-1b are converted to amines 2-4a and2-4b respectively. According to the method shown in Scheme 2, Turmericoil (containing ketones 2-1a and 2-1b and other molecules occurring inTurmeric) is reacted with amine 2-2 [wherein R^(3b) can be alkyl (e.g.isobutyl) or the like] to form compounds 2-3a and 2-3b respectively.Then, the resultant compounds 2-3a and 2-3b are then reduced, with areducing agent such as a borohydride, to from the nitrogen-containingcompounds 2-4a and 2-4b respectively (the reaction crude product alsoincludes other chemical compounds).

In the next step of the method, amines 2-4a and 2-4b areisolated/purified from the extract (the crude reaction product of the2-step reductive amination process). The conditioned extracts can befractionated by flash chromatography. The fractions that contain amines2-4a or 2-4b can undergo further purification/isolation according to themethods known to those in the art. Further isolation andcharacterization of the fraction that contains amines 2-4a or 2-4b mayfollow. In some embodiments, amine 2-4a can be subject to furtherseparation (such as using column chromatography) to isolate each of thediastereoisomers. In some embodiments, amine 2-4b can be subject tofurther separation (such as using column chromatography) to isolate eachof the diastereoisomers. The isolated amines 2-4a and 2-4b are testedfor their biological activities such as by those methods describedhereinwith.

Some examples of amine 2-2 used in the chemical conditioning process ofthe invention shown in Scheme 2 include akylamines such as methylamine,ethylamine, n-propylamine, isopropylamine, n-butylamine, 2-butylamine,isobutylamine, and tert-butylamine; phenylamine, and benzylamine.

In another embodiment, a specific example of the chemical conditioningprocess is shown in Scheme 3 below. Scheme 3 shows a reductive chemicalconditioning protocol (or a chemical conditioning protocol of reduction)performed on Turmeric oil in accordance with one embodiment of themethod, wherein Turmeric oil comprising ketones 3-1a and 3-1b arereduced/converted to alcohols 3-2a and 3-2b respectively. In someembodiments, alcohol 3-2a can be subject to further separation (such asusing column chromatography) to isolate each of the diastereoisomers. Insome embodiments, alcohol 3-2b can be subject to further separation(such as using column chromatography) to isolate each of thediastereoisomers. The alcohol derivatives 3-2a and 3-2b are tested fortheir biological activities such as by those methods describedhereinwith.

In some embodiments, the derivatives of Turmeric oil such as aminederivatives 2-4a and 2-4b and alcohol derivatives 3-2a and 3-2b possessbeta-secretase inhibitory activity, and/or inhibit amyloid production,amyloid assembly, the activity/effect of Abeta oligomers on neurons(such as neurons in the brain), amyloid aggregation, amyloid (includingamyloid oligomer) binding, or amyloid deposition. These compounds areuseful therapeutic agents for the treatment and prevention of cognitivedecline, amyloid production, neurodegeneration, and Alzheimer's disease.

New lead compounds generated by this chemical conditioning method canalso be prepared by the synthetic methods described hereinwith.

Synthesis

Compounds of embodiments the invention, including salts thereof, can beprepared using known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., Wiley &Sons, Inc., New York (1999), which is incorporated herein by referencein its entirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC).

The compounds of embodiments of the invention can be prepared, forexample, according to the reaction pathways, synthetic procedures, andtechniques described below.

As shown in Scheme 4, ketone 4-1 can be reacted with 1,3-diester 4-2[wherein each R¹⁰ can be independently alkyl (e.g. methyl) or the like]in the presence of either an acid or a base catalyst (through anenolate), followed by hydrolysis (for example under acidic condition)and loss of CO₂, to afford acid 4-3. Reaction of acid 4-3 (or its estersuch as methyl ester) with a reducing reagent such as LAH, followed byoxidation of the intermediate alcohol with an oxidation reagent such asthe Dess-Martin periodinane, can afford aldehyde 4-4. Reaction ofaldehyde 4-4 with an organometallic compound such as an organo lithiumcompound 4-5 can form alcohol 4-6. Different diastereomers of alcohol4-6 can be separated by methods known to those skilled in the art suchas column chromatography. See e.g. A. Li, et. al, “Total asymmetricsynthesis of (7S,9R)-(+)-bisacumol”, Tetrahedron: Asymmetry (2003),14(1), 75-78. Oxidation of alcohol 4-6 with a suitable oxidation reagentsuch as MnO₂ can afford ketone 4-7. Reductive amination of ketone 4-7with a suitable amine R^(3b)NH₂ in the presence of a suitable hydridesuch as sodium borohydride can afford amine 4-8. Different diastereomersof amine 4-8 can be separated by methods known to those skilled in theart such as column chromatography.

As shown in Scheme 4a, aromatic compound 4a-0-1 can be reduced tocyclohexa-1,4-diene 4a-02 under Birch reduction conditions. See e.g.Rabideau, P. W., “The metal-ammonia reduction of aromatic compounds”,Tetrahedron, Volume 45, Issue 6, 1989, pages 1579-1603. Under acidicconditions (such as in the presence of catalytic amount of HCl or aceticacid), cyclohexa-1,4-diene 4a-02 can rearrange to the thermodynamicallymore stable cyclohexa-1,3-diene 4a-1. Cyclohexa-1,3-diene 4a-1. can beconverted to alcohol 4a-6 or amine 4a-8 according to methods similar tothose described in Scheme 4.

As shown Scheme 5, treatment of styrene derivative 5-1 with AD-mix-α(See e.g. Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;et al. J. Org. Chem. 1992, 57, 2771) affords diol 5-2. See A. Li, et.al, “Total asymmetric synthesis of (7S,9R)-(+)-bisacumol”, Tetrahedron:Asymmetry (2003), 14(1), 75-78. Stereo-selective reduction of thebenzylic OH of diol 5-2 with Raney nickel gives alcohol 5-3. See id.Treament of alchol 5-3 with PPh₃ and CBr₄ in a suitable solvent such asmethylene chloride affords bromide 5-4. Conversion of bromide 5-4 to thecorresponding Grignard reagent in the presence of magnesium powder andCH₃I (by metal-halogen exchange), followed by reaction with 3-methylcrotonaldehyde, provides alcohol 5-5. Different diastereomers of alcohol5-5 can be separated by methods known to those skilled in the art suchas column chromatography. See id. Alcohol 5-5 can be transformed intoits corresponding amine compound 5-6 using similar methods to thoseoutlined in Scheme 4.

Those skilled in the art can recognize that in all of the schemesdescribed herein, if there are functional (reactive) groups present on asubstituent group such as R¹, R², R³, and R⁴, etc., further modificationcan be made if appropriate and/or desired. For example, an OH group canbe converted into a better leaving group such as mesylate, which in turnis suitable for nucleophilic substitution, such as by Br. Thus, acompound of Formula I (such as compound 4-8 of Scheme 4) having asubstituent which contains a functional group can be converted toanother compound of Formula I having a different substituent group.

As used herein, the term “reacting” refers to the bringing together ofdesignated chemical reactants such that a chemical transformation takesplace generating a compound different from any initially introduced intothe system. Reacting can take place in the presence or absence ofsolvent.

Methods

In some embodiments, the compounds of present invention inhibit, treat,or abate (partially inhibit) binding of amyloid (including Abetaoligomers) to neurons (such as neurons in the brain) and are useful forthe inhibition, treatment, and abatement of cognitive decline and/orAlzheimer's disease. In some embodiments, the compounds of presentinvention inhibit, treat, or abate (partially inhibit) one or more ofamyloid aggregation, amyloid oligomer binding, and amyloid deposition.In some embodiments, the compounds of present invention inhibit, treat,or abate (partially inhibit) amyloid deposition. In some embodiments,the compounds of present invention inhibit, treat, or abate (partiallyinhibit) the activity/effect of Abeta oligomers on neurons (such asneurons in the brain) and are useful for the inhibition, treatment, andabatement of cognitive decline and/or Alzheimer's disease. In someembodiments, the compounds of present invention inhibit, treat, or abate(partially inhibit) the activity/effect of Abeta oligomers on neurons(such as neurons in the brain) via disruption of Abeta oligomers,inhibition of Abeta oligomer binding to neurons, and/or counteraction ofsignal transduction mechanisms of action initiated by Abeta oligomerbinding.

In some embodiments, the compounds show activity in a beta-secretaseassay and are useful for the inhibition, treatment, and abatement ofcognitive decline and Alzheimer's disease. In some embodiments thederivative of ginger oil is a compound in purified and isolated form(for example, with a purity of greater than 80%, 85%, 90%, 95%, 98%, or99% by weight). The compounds and methods described herein may be usedto treat one or more symptoms of cognitive decline and/or Alzheimer'sdisease such as memory loss, confusion, impaired judgment, personalitychanges, disorientation, and loss of language skills. Further, thecompounds and methods described herein may be useful in inhibiting,treating, and/or abating cognitive decline and/or Alzheimer's disease byrestoring long term potentiation, and/or inhibiting, treating, orabatement of one or both of neurodegeneration and general amyloidosis,more specifically, by inhibiting, treating, or abatement of one or moreof amyloid production, amyloid assembly, amyloid aggregation, amyloid(including amyloid oligomer) binding, and amyloid deposition.

In some embodiments, compounds of the invention can inhibit, treat, orabate one or more of amyloid production, amyloid assembly, amyloidaggregation, amyloid oligomer binding, and amyloid deposition. In someembodiments, compounds of the invention can restore long termpotentiation, inhibit, treat, or abate one or both of neurodegenerationand general amyloidosis.

In some embodiments, compounds of present invention inhibit, treat, orabate (partially inhibit) one or more of amyloid aggregation, amyloidoligomer binding, and amyloid deposition. In some embodiments, thecompounds of present invention inhibit (or partially inhibit) amyloiddeposition. In some embodiments, the compounds of present inventioninhibit, treat, or abate (partially inhibit) binding of amyloid(including Abeta oligomers) to neurons (such as neurons in the brain).In some embodiments, the compounds of present invention are useful forthe inhibition, treatment, and abatement of cognitive decline and/orAlzheimer's disease.

In some embodiments, compounds of the invention can inhibit activity ofbeta-secretase. In some embodiments, compounds of the invention can beused in methods of inhibiting activity of beta-secretase by contactingthe beta-secretase with any one or more of the compounds or compositionsdescribed herein.

Another aspect of the present invention pertains to methods of treatingcognitive decline and/or Alzheimer's disease in an individual (e.g.,patient) by administering to the individual a therapeutically effectiveamount or dose of a compound of the present invention or apharmaceutical composition thereof.

Treatment of the diseases/disorders herein includes treating one or moresymptoms associated with the diseases/disorders, for example, symptomsof cognitive decline and/or Alzheimer's disease.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a beta-secretase or a neuron cell (or a neuroncell in the presence of one or more of beta-amyloid oligomers) with acompound of the invention includes the administration of a compound ofthe present invention to an individual or patient, such as a human,having a beta-secretase or a neuron cell, as well as, for example,introducing a compound of the invention into a sample containing acellular or purified preparation containing the a beta-secretase or aneuron cell (or a neuron cell in the presence of one or more ofbeta-amyloid oligomers).

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting/retardingthe disease; for example, inhibiting/retarding a disease, condition ordisorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder; and(3) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., reversing the pathology and/or symptomatology) such as decreasingthe severity of disease or completely eliminating/curing the disease. Asused herein, treating a disease further includes treating one or moresymptoms associated with the disease.

Combination Therapies

In certain embodiments, one or more additional pharmaceutical agents fortreatment of cognitive decline and/or Alzheimer's disease can be used incombination with the compounds of the present invention for treatment ofcognitive decline and/or Alzheimer's disease. The one or more additionalpharmaceutical agents can be administered to a patient simultaneously orsequentially.

Pharmaceutical Formulations and Dosage Forms

In certain embodiments, the compounds of the invention can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Embodiments of this invention also include pharmaceutical compositionswhich contain, as the active ingredient, one or more of the compounds ofthe invention above in combination with one or more pharmaceuticallyacceptable carriers (excipients). In making the compositions of theinvention, the active ingredient is typically mixed with an excipient,diluted by an excipient or enclosed within such a carrier in the formof, for example, a capsule, sachet, paper, or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art, for example see InternationalPatent Application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The active compound can be effective over a wide dosage range and can begenerally administered in a pharmaceutically effective amount. Forexample, the dosage of the active compounds of the invention as employedfor the treatment of a patient in need thereof (such as an adult human)may range from 0.1 to 3000 mg per day, depending on the route andfrequency of administration. Such a dosage corresponds to 0.001 to 50mg/kg per day. In some embodiments, the dosage of the active compoundsof the invention as employed for the treatment of a patient in needthereof (such as an adult human) may range from 1 to 2000 mg per day,from 1 to 1000 mg per day, from 10 to 1000 mg per day, or from 10 to 500mg per day. It will be understood, however, that the amount of thecompound actually administered will usually be determined by aphysician, according to the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical excipient to form a solidpre-formulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepre-formulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid pre-formulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention canvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. The proportion or concentration of a compound of theinvention in a pharmaceutical composition can vary depending upon anumber of factors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, thecompounds of the invention can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral administration. Some typical dose ranges are from about 1mg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled compounds ofthe invention (radio-labeled, fluorescent-labeled, etc.) that would beuseful not only in radio-imaging but also in assays, both in vitro andin vivo, for localizing and quantitating the enzyme in tissue samples,including human, and for identifying ligands by inhibition binding of alabeled compound. Accordingly, the present invention includes enzymeassays that contain such labeled compounds.

Embodiments of the present invention further includesisotopically-labeled compounds of the invention. An “isotopically” or“radio-labeled” compound is a compound of the invention where one ormore atoms are replaced or substituted by an atom having an atomic massor mass number different from the atomic mass or mass number typicallyfound in nature (i.e., naturally occurring). Suitable radionuclides thatmay be incorporated in compounds of the present invention include butare not limited to ²H (also written as D for deuterium), ³H (alsowritten as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F,³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. Theradionuclide that is incorporated in the radio-labeled compounds willdepend on the specific application of that radio-labeled compound. Forexample, for in vitro receptor labeling and competition assays,compounds that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S, or willgenerally be most useful. For radio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I,¹²³I, ¹²⁴I ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generally be most useful.

It is understood that a “radio-labeled compound” is a compound that hasincorporated at least one radionuclide. In some embodiments theradionuclide is selected from ³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br.

In some embodiments, the labeled compounds of the present inventioncontain a fluorescent label.

Synthetic methods for incorporating radio-isotopes and fluorescentlabels into organic compounds are well known in the art.

A labeled compound of the invention (radio-labeled, fluorescent-labeled,etc.) can be used in a screening assay to identify/evaluate compounds.For example, a newly synthesized or identified compound (i.e., testcompound) which is labeled can be evaluated for its ability to bind abeta-secretase or a neuron cell (or a neuron cell in the presence of oneor more of beta-amyloid oligomers) by monitoring its concentrationvariation when contacting with the beta-secretase or the neuron cell (orthe neuron cell in the presence of one or more of beta-amyloidoligomers), through tracking the labeling. For another example, a testcompound (labeled) can be evaluated for its ability to reduce binding ofanother compound which is known to bind to beta-secretase or neuron cell(i.e., standard compound). Accordingly, the ability of a test compoundto compete with the standard compound for binding to the beta-secretaseor the neuron cell directly correlates to its binding affinity.Conversely, in some other screening assays, the standard compound islabeled and test compounds are unlabeled. Accordingly, the concentrationof the labeled standard compound is monitored in order to evaluate thecompetition between the standard compound and the test compound, and therelative binding affinity of the test compound is thus ascertained.

Kits

Embodiments of the present invention also includes pharmaceutical kitsuseful, for example, in the treatment or prevention of cognitive declineand/or Alzheimer's disease which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of the invention. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results. Certain compounds of the Examples were found to beinhibit, treat, or abate one or more of amyloid production, amyloidassembly, the activity/effect of Abeta oligomers on neurons (such asneurons in the brain), amyloid aggregation, amyloid oligomer binding,and amyloid deposition according to one or more of the assays providedherein. In some further embodiments, certain compounds of the Exampleswere found to be inhibit, treat, or abate one or more of theactivity/effect of Abeta oligomers on neurons (such as neurons in thebrain), amyloid aggregation, amyloid (including amyloid oligomer)binding, and amyloid deposition according to one or more of the assaysprovided herein.

In some embodiments, the compound of invention has an IC₅₀ value of lessthan 100 μM, 50 μM, 20 μM, 15 μM, 10 μM, 5 μM, 1 μM, 500 nM, 100 nM, 50nM, or 10 nM with respect to inhibition of one or more of theactivity/effect of Abeta oligomers on neurons (such as neurons in thebrain), amyloid aggregation, amyloid (including amyloid oligomer)binding, and amyloid deposition. In some embodiments, the compound ofinvention has an IC₅₀ value of less than 100 μM, 50 μM, 20 μM, 15 μM, 10μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, or 10 nM with respect toinhibition the activity/effect of Abeta oligomers on neurons (such asneurons in the brain).

In some embodiments, percentage inhibition of the compound of inventionto one or more of the activity/effect of Abeta oligomers on neurons(such as neurons in the brain), amyloid aggregation, amyloid (includingamyloid oligomer) binding, and amyloid deposition was measured at aconcentration of from 10 nM to 10 μM. In some embodiments, thepercentage inhibition measured is about 1% to about 20%, about 20% toabout 50%, about 1% to about 50%, or about 1% to about 80%.

The invention may be appreciated in certain aspects with reference tothe following examples, offered by way of illustration, not by way oflimitation. Materials, reagents and the like to which reference is madein the following examples are obtainable from commercial sources, unlessotherwise noted.

EXAMPLES Materials And Methods Turmeric Oil

The light oil extract from turmeric was obtained by supercritical CO₂extraction.

Example 1 Conditioned Extraction of Turmeric Oil (Reductive Amination)Reaction of Turmeric Oil with Isobutylamine Followed by Reduction withSodium Borohydride in Methanol and by Fractioning Using ColumnChromatography

Turmeric oil (10 g) was dissolved in methanol (250 mL) and isobutylamine(4.0 mL) was added. The mixture was maintained at room temperature for16 hours. At this time the reaction mixture was cooled to 0° C. on anice bath. A solution of sodium borohydride (5 g) in methanol (50 mL) wasadded portion-wise over 30 minutes with vigorous stirring. After theaddition was complete the resultant mixture was maintained at reflux for16 hours. At this time the reaction mixture was cooled to roomtemperature and poured into saturated aqueous sodium bicarbonatesolution (300 mL). The resulting mixture was concentrated by rotaryevaporation and the aqueous residue was partitioned between water andchloroform. The chloroform layer was dried over anhydrous sodium sulfateand then filtered and concentrated. The crude product was thenfractionated using silica gel column chromatography employing a gradientfrom 100% chloroform to chloroform:methanol (4:1). Twenty combinedfractions from relatively non-polar to polar were collected andconcentrated. Each fraction was submitted for biological testing. Theactive product-containing fractions were the relatively polar fractions.

An active product-containing fraction (Fraction 1A) was subject tofurther separation by column chromatography and two major componentswere isolated: Component 1A-1 and Component 1A-2.

The weight ratio of Turmeric oil to isobutylamine used in the reductiveamination is about 3:1 (from 2 to 4:1).

Purity Determination

The purity of 1 A was measure by HPLC. Only two major peaks (twocomponents) were present. The HPLC conditions used are as follows.

HPLC Conditions

Mobile Phase A: 13.3 mM ammonium formate/6.7 mM formic acid in water.

Mobile Phase B: 6 mM ammonium formate/3 mM formic acid in water/CH₃CN(1/9, v/v)

Column 1: Synergi Fusion-RP 100A Mercury, 2×20 mm, 2.5 micron(Phenomenex Part No 00M-4423-B0_CE)

Column 2: Synergi Max-RP 80, 2×50 mm, 4 micron zPhenomenex Part No00B-4337-B0)

Gradient Program: (the same for both column I and II) Time, minute %Phase B Flow rate, ml/min 0 20 0.5 1 20 0.5 2.5 100 0.5 3.4 100 0.5 3.520 0.5 4.5 20 0.5 RT on Column I RT on Column I Component Number(minute) (minute) 1A-1 2.15 2.46 1A-2 2.24 2.55

Component 1A-1: ¹H NMR (500 MHz, CDCl3) δ 7.11, 7.09, 5.23, 3.72, 2.94,2.51, 2.34, 2.31, 1.92, 1.72, 1.71, 1.58, 1.29, 1.27, 0.92. ¹³C NMR (125MHz, CDCl3) 144.50, 135.37, 135.03, 129.03, 126.89, 126.70, 66.97,49.25, 46.39, 35.05, 32.46, 25.83, 20.96, 20.67, and 18.36. MS (M+H⁺)m/z 274.3.

The structure of Fraction 1A, Component 1A-1 is determined to be asfollows.

Component 1A-2: ¹H NMR (500 MHz, CDCl3) δ 5.77, 5.65, 5.45, 5.23, 3.96,2.94, 2.93, 2.51, 2.31, 2.30, 2.05, 1.92, 1.72, 1.71, 1.58, 1.29, 1.27,0.92. ^(D)C NMR (125 MHz, CDCl3) δ 144.50, 135.37, 130.96, 127.86,126.70 120.80, 66.72, 49.25, 46.39, 35.05, 32.46, 25.83, 21.79, 21.79,20.67, and 18.36. MS (M+H⁺) m/z 276.3.

The structure of Fraction 1A, Component 1A-2 is determined to be asfollows.

The chemical shift measure by ¹H NMR may vary, for example, up to 0.3ppm. The chemical shift measure by ¹³H NMR may vary, for example, up to0.6 ppm. The analytical Mass Spectrum may have an experimental error of+/−0.4.

Example 2 Conditioned Extraction of Turmeric Oil (Reduction) Reductionof Turmeric Oil with Sodium Borohydride in Methanol and by FractioningUsing Column Chromatography

Turmeric oil (10 g) was dissolved in methanol (250 mL). The reactionmixture was cooled to 0° C. on an ice bath. A solution of sodiumborohydride (5 g) in methanol (50 mL) was added portion-wise over 30minutes with vigorous stirring. When the addition was complete themixture was maintained at reflux for 16 hr. At this time the reactionmixture was cooled to room temperature and poured into saturated aqueoussodium bicarbonate (300 mL). The resulting mixture was concentrated byrotary evaporation and the aqueous residue was partitioned between waterand chloroform. The chloroform layer was dried over anhydrous sodiumsulfate and then filtered and concentrated. The product was thenfractionated using silica gel column chromatography employing a gradientfrom 100% chloroform to chloroform:methanol (4:1). Twenty combinedfractions from relatively non-polar to polar were collected andconcentrated. Each fraction was submitted for biological testing. Theactive product-containing fractions were the relatively polar fractions.

The molar ratio of sodium borohydride to Turmeric oil (the ketones andaldehydes therein) is greater than 1:1, 2:1, 3:1, 4:1, 5:1, or 6:1 toensure that reduction of ketones and aldehyes to alcohols. In someembodiments, the weight ratio of sodium borohydride to Turmeric oil isabout 0.5:1 (from 0.3:1 to about 0.8:1).

An active product-containing fraction (Fraction 2A) was subject tofurther separation by column chromatography and two major componentswere isolated: Component 2A-1 and Component 2A-2.

Component 2A-1: ¹H NMR (500 MHz, CDCl3) δ 7.11, 7.09, 5.17, 4.45, 2.32,2.76 1.75, 1.58, 1.52, and 1.26. ¹³C NMR (125 MHz, CDCl3) δ 144.3,135.37, 135.03, 129.11, 128.32, 126.90, 67.10, 42.05, 39.64, 25.81,23.00, 21.01, and 18.12. MS (M+H¹) m/z 219.2.

The structure of Fraction 2A, Component 2A-1 is determined to be asfollows.

Component 2A-2: ¹H NMR (500 MHz, CDCl3) δ 6.18, 5.70, 5.56, 5.17, 4.24,2.32, 2.80, 2.29, 2.15, 1.95, 1.68, 1.75, 1.58, 1.42, 1.27. ¹³C NMR δ144.10, 135.00, 134.10, 129.58, 128.32, 125.3, 66.90, 46.18, 41.17,39.55, 37.14, 25.81, 24.50, 23.00, and 18.12. MS (M+H⁺) m/z 221.1.

The structure of Fraction 2A, Component 2A-2 is determined to be asfollows.

The chemical shift measure by ¹H NMR may vary, for example, up to 0.3ppm. The chemical shift measure by ¹³H NMR may vary, for example, up to0.6 ppm. The analytical Mass Spectrum may have an experimental error of+/−0.4.

Purity Determination

The purity of Fraction 2A was measure by HPLC. Only two major peaks (twocomponents) were present.

Example AA Exocytosis Assay/MTT Assay

Primary neurons from E18 Sprague-Dawley rat embryos are plated atoptimized concentrations in 384 well plates in NB media (Invitrogen).Neurons are maintained in cultures for 3 weeks, with twice weeklyfeeding of NB media with N₂ supplement (Invitrogen). A test compound isadded to cells, followed by addition of Vehicle or Abeta oligomerpreparations (1.5 μM), and incubated for 1 to 24 hr at 37° C. in 5% CO₂.MTT reagent (3-(4,5-dimethylthizaol-2yl)-2,5diphenyl tetrazoliumbromide) (Roche Molecular Biochemicals) is reconstituted in phosphatebuffered saline to 5 mg/mL. 10 μL of MTT labeling reagent is added toeach well and incubated at 37° C. for 1 h, then imaged.

Each assay plate is formatted so that compounds are tested with andwithout Abeta on each plate. This design eliminates toxic ormetabolically active compounds early on in the screening cascade (at thelevel of the primary screen).

Similar procedures for exocytosis assays/MTT assays can be found in theliterature. See e.g., Liu Y, et. al., Detecting bioactive amyloid betapeptide species in Alzheimer's disease. J Neurochem. 2004 November;91(3):648-56; Liu Y, and Schubert D. “Cytotoxic amyloid peptides inhibitcellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reduction by enhancing MTT formazan exocytosis.” J Neurochem. 1997December; 69(6):2285-93; and Liu Y, and Schubert D. “TreatingAlzheimer's disease by inactivating bioactive amyloid beta peptide”Curr. Alzheimer Res. 2006 April; 3(2):129-35.

Experimental Controls

Abeta 1-42 oligomers made according to published methods methods [Seee.g. Dahlgren et al., “Oligomeric and fibrillar species of amyloid-betapeptides differentially affect neuronal viability” J Biol Chem. 2002Aug. 30; 277(35):32046-53. Epub 2002 Jun. 10.; LeVine H 3rd.“Alzheimer's beta-peptide oligomer formation at physiologicconcentrations” Anal Biochem. 2004 Dec. 1; 335(1):81-90; Shrestha et.al, “Amyloid beta peptide adversely affects spine number and motility inhippocampal neurons” Mol Cell Neurosci. 2006 November; 33(3):274-82.Epub 2006 Sep. 8; Puzzo et al., “Amyloid-beta peptide inhibitsactivation of the nitric oxide/cGMP/cAMP-responsive element-bindingprotein pathway during hippocampal synaptic plasticity” J Neurosci. 2005Jul. 20; 25(29):6887-97; Barghorn et al., “Globular amyloid beta-peptideoligomer—a homogenous and stable neuropathological protein inAlzheimer's disease” J Neurochem. 2005 November; 95(3):834-47. Epub 2005Aug. 31; Johansson et al., Physiochemical characterization of theAlzheimer's disease-related peptides A beta 1-42 Arctic and A beta 1-42wt. FEBS J. 2006 June; 2 73(12):2618-30] as well as brain-derived Abetaoligomers (See e.g. Walsh et al., Naturally secreted oligomers ofamyloid beta protein potently inhibit hippocampal long-term potentiationin vivo. Nature (2002). 416, 535-539; Lesne et al., A specificamyloid-beta protein assembly in the brain impairs memory. Nature. 2006Mar. 16; 440(7082):352-7; Shankar et al, Amyloid-beta protein dimersisolated directly from Alzheimer's brains impair synaptic plasticity andmemory. Nat Med. 2008 August; 14(8):837-42. Epub 2008 Jun. 22)constitute the positive controls. Negative controls includevehicle-treated neurons as well as neurons treated with 28 μMconcentrations of memantine. Memantine produces 50% inhibition ofoligomer effects at this dose. These controls, on each plate, serve asnormalization tools to calibrate assay performance on a plate-by-platebasis.

Primary Neuronal Cultures

Optimal cell density is determined based on cellular response to Abetaoligomers using the exocytosis assay as a readout, andimmunohistochemical analysis of the relative proportion of glia toneurons in the cultures. Cultures are monitored on a weekly basis withimmunohistochemistry and image processing-based quantification tomonitor the percentage of the cultures that are neurons vs. glia (Glialcells). Cultures containing more than 20% glia (positive for GFAP) vs.neurons (staining positively with antibodies directed against MAP2) atthe screening age of 21 days in vitro (21 DIV) are rejected.

Abeta Oligomer Preparations

Human amyloid peptide 1-42 is obtained from California Peptide, withlot-choice contingent upon quality control analysis. Quality controls ofoligomer preparations consist of Westerns to determine oligomer sizeranges and relative concentrations, and the MTT assay to confirmexocytosis acceleration without toxicity. Toxicity is monitored in eachimage-based assay via quantification of nuclear morphology visualizedwith the DNA binding dye DAPI (Invitrogen). Nuclei that are fragmentedare considered to be in late stage apoptosis (Majno and Joris '95).Peptide lots producing unusual peptide size ranges or significanttoxicity at a standard 1.5 uM concentration on neurons are rejected.Plate-based controls—The assay optimization will be complete whenreformatted plates achieve a minimum of statistically significanttwo-fold separation between vehicle and Abeta oligomer-treated neurons(p<0.01, Student's t-test, unequal variance) on a routine basis, with nomore than 10% CV between plates, equivalent to its current performance.

Statistical Software and Analysis:

Data handling and analysis are accomplished by Cellomics VTI imageanalysis software and STORE automated database software. Because of thelow dynamic range and neuronal well-to-well variability after threeweeks in culture, statistical comparisons are made via pairwiseTukey-Kramer analysis to determine the significance of the separationbetween compound+Abeta oligomers from Abeta alone, and between compoundalone from vehicle. These statistics are more akin to what is seen inanimal behavioral testing than the z′ statistic that has been used forthe past two decades in high throughput screening. The ability of matureprimary neurons to more closely approximate the electrophysiologicallymediated signal transduction network of the adult brain justifies thisscreening strategy. Power analysis will be set for a number of replicatescreening wells that will minimize false negatives (e.g N=4) and shiftthe burden of distinguishing false positives from actual hits todose-response confirmation screening. Rank ordering of compounds is doneon the basis of secondary assay mechanism of action and physicochemicalproperties of the compound structures. Certain test compoundssignificantly reverse the effects of Abeta oligomers but not affectneuronal metabolism.

Fraction 1A was dosed in the MTT assay and was shown to block the Abetaoligomer-induced acceleration of exocytosis with an EC₅₀ of 10.5 μM,indicating that one or both of Component 1A-1 and Component 1A-2block/abate the activity/effect of Abeta oligomer on neuron cells.

Fraction 2A was dosed in the MTT assay and was shown to block the Abetaoligomer-induced acceleration of exocytosis with an EC₅₀ of 25.4 μM,indicating that one or both of Component 2A-1 and Component 2A-2block/abate the activity/effect of Abeta oligomer on neuron cells.

Example BB Binding Assay

Each test compound was added to a plate followed by an addition of oneor more of Abeta 1-42 Oligomers. The plates were fixed with 3.7%paraformaldehyde in phosphate buffered saline (PBS) for 15 min. Theplate was then washed 3× with PBS for 5 min each. The plates wereblocked at room temperature for 1 hour in 5% goat serum and 0.5% TritonX-100 (CAS number: 9002-93-1) in PBS. Primary antibodies (anti-MAP 2polyclonal, Millipore #AB5622 and anti-Beta Amyloid 6E10 monoclonal,Convance #SIG-39300) were diluted 1:1000 in 5% goat serum with PBS.Primary antibodies were incubated either overnight at 4° C. or 1 hour atRT. The plate was then washed 3× with PBS for 5 min each. Secondaryantibodies (Alex Flor 488 polyclonal, Invitrogen # A11008 and Alexa Flor647 monoclonal, Invitrogen #A21235) were diluted 1:1000 in 5% goat serumwith PBS. Secondary antibodies were incubated at RT for 1 hr. The plateswere washed once with PBS. DAPI (4′,6-diamidino-2-phenylindole,Invitrogen) was then applied at 0.03 μg/μL and incubated at RT for 5min, then washed with PBS. Image process was carried out for analysis.

Similar procedures for binding assays can be found in the literature.See e.g., Look G C, et. al. Discovery of ADDL—targeting small moleculedrugs for Alzheimer's disease. Curr Alzheimer Res. 2007 December;4(5):562-7. Review.

The EC₅₀ of Fraction 2A was determined to be 14.5 μM according thebinding assay.

Abeta oligomer preparations Human amyloid peptide 1-42 is obtained fromCalifornia Peptide, with lot-choice contingent upon quality controlanalysis. Abeta 1-42 oligomers made according to published methods [Seee.g. Dahlgren et al., “Oligomeric and fibrillar species of amyloid-betapeptides differentially affect neuronal viability” J Biol Chem. 2002Aug. 30; 277(35):32046-53. Epub 2002 Jun. 10.; LeVine H 3rd.“Alzheimer's beta-peptide oligomer formation at physiologicconcentrations” Anal Biochem. 2004 Dec. 1; 335(1):81-90; Shrestha et.al, “Amyloid beta peptide adversely affects spine number and motility inhippocampal neurons” Mol Cell Neurosci. 2006 November; 33(3):274-82.Epub 2006 Sep. 8; Puzzo et al., “Amyloid-beta peptide inhibitsactivation of the nitric oxide/cGMP/cAMP-responsive element-bindingprotein pathway during hippocampal synaptic plasticity” J Neurosci. 2005Jul. 20; 25(29):6887-97; Barghorn et al., “Globular amyloid beta-peptideoligomer—a homogenous and stable neuropathological protein inAlzheimer's disease” J Neurochem. 2005 November; 95(3):834-47. Epub 2005Aug. 31; Johansson et al., Physiochemical characterization of theAlzheimer's disease-related peptides A beta 1-42 Arctic and A beta 1-42wt. FEBS J. 2006 June; 2 73(12):2618-30] as well as brain-derived Abetaoligomers (See e.g. Walsh et al., Naturally secreted oligomers ofamyloid beta protein potently inhibit hippocampal long-term potentiationin vivo. Nature (2002). 416, 535-539; Lesne et al., A specificamyloid-beta protein assembly in the brain impairs memory. Nature. 2006Mar. 16; 440(7082):352-7; Shankar et al, Amyloid-beta protein dimersisolated directly from Alzheimer's brains impair synaptic plasticity andmemory. Nat Med. 2008 August; 14(8):837-42. Epub 2008 Jun. 22) willserve as positive controls. Quality controls of oligomer preparationsconsist of Westerns to determine oligomer size ranges and relativeconcentrations, and the MTT assay to confirm exocytosis accelerationwithout toxicity. Toxicity is monitored in each image-based assay viaquantification of nuclear morphology visualized with the DNA binding dyeDAPI (Invitrogen). Nuclei that are fragmented are considered to be inlate stage apoptosis (Majno and Joris Apoptosis, oncosis, and necrosis.An overview of cell death. Am J Pathol 1995; 146:3-16). Peptide lotsproducing unusual peptide size ranges or significant toxicity atstandard concentrations on neurons are rejected.

Image Processing

Images were captured and analyzed with the Cellomics VTI automatedmicroscope platform, using the Neuronal Profiling algorithm. Forstatistical analysis, a Tukey-Kramer pair-wise comparison with unequalvariance was used.

Western Blots

Samples containing Abeta 1-42 were diluted (1:5) in non-reducing lanemarker sample buffer (Pierce #1859594). A 30 microliter (μL) sample wasloaded onto an eighteen well precast 4-15% Tris-HCl gel (BIORAD#345-0028). Electrophoresis was performed in a BIO-RAD Criterian precastgel system using Tris-Glycine buffer at 125 volt (V) for 90 minutes. Thegels were blotted onto 0.2 μM nitrocellulose membranes inTris-Glycine/10% methanol buffer at 30V for 120 minutes. The membraneswere boiled for 5 minutes in a PBS solution and blocked over night withTBS/5% milk solution at 4° C. The membrane was probed with 6E10-HRP(Covance #SIG-39345) diluted to 10 μg/mL in TBS/1% milk solution for onehour at room temperature. Membrane was washed three times for 40 minuteseach with a solution of TBS/0.05% tween-20 and developed with ECLreagent (BIO-RAD #162-0112) for 5 minutes. Image acquisition wasperformed on an Alpha Innotech FluorChem Q quantitative imaging systemand analyzed with AlphaView Q software.

Fraction 1A was shown to partially block binding of the Abeta oligomerligand to neurons by 33% according to the binding assay (using imagingprocessing algorithm)..

Fraction 2A was shown to partially block binding of the Abeta oligomerligand to neurons by 35% according to the binding assay (using imagingprocessing algorithm)..

PK Studies:

PK studies are performed at CEREP Inc of Redmond Wash., according totheir standard protocols: The plasma samples were processed usingacetonitrile precipitation and analyzed by HPLC-MS or HPLC-MS/MS. Peakareas were recorded, and the concentrations of the test compound in theunknown plasma samples were determined using the respective calibrationcurve. The reportable linear range of the assay was determined, alongwith the lower limit of quantitation (LLQ).

NMR Spectroscopy and Mass Spectrometry:

Active fractions were analyzed by 1H NMR (Varian 500 MHz NMRspectrometer) and purified compounds were characterized using acombination 1D and 2D 1H NMR experiments and 13C NMR experiments.Structure proof was obtained using these NMR techniques in combinationwith low resolution mass spectrometry to determine molecular weight andhigh resolution mass spectrometry (Thermo Finnigan LCQ Ion trap) todetermine composition-of-matter.

Example CC Pharmacokinetic Studies

Pharmacokinetic studies were performed according to the followingprotocols: The plasma samples were processed using acetonitrileprecipitation and analyzed by HPLC-MS or HPLC-MS/MS. Peak areas wererecorded, and the concentrations of the test compound in the unknownplasma samples were determined using the respective calibration curve.The reportable linear range of the assay was determined, along with thelower limit of quantitation (LLQ). For example, Component 1A-1 wasdetermined to have a half life of 50 minutes in the plasma of rats wheninjected intravenously at 1 mg/Kg; Component 1A-2 was determined to havea half life of 70 minutes in the plasma of rats when injectedintravenously at 1 mg/Kg; and Component 2A-1 was determined to have ahalf life of 180 minutes in the plasma of rats when injectedintravenously at 1 mg/Kg. However, the experimental condition used didnot give a detectable half lift for Component 2A-2.

Example DD Abeta Oligomer Formation Inhibition Assay

Abeta 42 oligomer formation can be readily assayed in a multiwell formatand used to determine the ability of a test compound to block theformation of soluble high-molecular weight (>20 kDa) oligomers. See e.g.assays described in Harry LeVine III, “Biotin-avidin interaction-basedscreening assay for Alzheimer's β-peptide oligomer inhibitors”,Analytical Biochemistry 356 (2006) 265-272.

100 μL, of different concentrations of a test compound in 50 mM NaP_(i),150 mM NaCl, and 0.02% (w/v) NaN₃ pH 7.5 was added to wells of a 96-wellplate containing 2 μL, of freshly HFIP(1,1,1,3,3,3-Hexafluoro-2-propanol)-depolymerized 2.5 μg/ml bio-Abeta42in DMSO, giving a total concentration of 50 ng/ml (11 nM) peptide and 2%(v/v) DMSO. After 30 min at room temperature, 50-μL aliquots weretransferred to an NA/SA-HRP (NeutrAvidin/secondary antibody andstreptaviding Horseradish peroxidase) single-site assay plate. 100 μL,of tetramethylbenzidine/H2O2 substrate solution is added, and the plateis incubated at room temperature for 2-30 min, depending on theconcentration of bio-Abeta 42 peptide in the assay. The OD_(450nm) isdetermined on a Biotech Synergy HT plate reader after stopping thereaction with 100 μL, of 1% (v/v) H₂SO₄. The ability of a test compoundto block the formation of soluble high-molecular weight (>20 kDa)oligomers is determined by the concentration of the bio-Abeta 42 peptideformed. See id.

According to the assay, Fraction 1A does not inhibit the formation ofAbeta oligomers. Therefore Fraction 1A likely inhibits Abeta oligomerbinding to neurons by acting directly on neuronal receptors to preventoligomer binding, or by causing Abeta oligomers to disassemble.

Example EE A Primary Neuron-Based Functional Screening Assay to DetectSmall Molecule Abeta Oligomer Blockers

Primary rat neurons grown for at least 3 weeks in vitro were chosen asthe basis for this screening assay. These neurons express the fullcomplement of synaptic proteins characteristic of neurons in the maturebrain, and exhibit a complex network of activity-dependent electricalsignaling. Neurons and glia in such cultures have molecular signalingnetworks exhibiting excellent registration with intact brain circuitry,and for this reason have been used for over two decades as a modelsystem for learning and memory (See e.g. Kaech S, Banker G. Culturinghippocampal neurons. Nat Protoc. 2006; 1(5):2406-15. Epub 2007 Jan 11;See also Craig A M, Graf E R, Linhoff M W. How to build a centralsynapse: clues from cell culture. Trends Neurosci. 2006 January;29(1):8-20. Epub 2005 Dec. 7. Review). More complex systems such asacute or organotypic brain slices are very useful but not amenable tohigh throughput screening. Immortalized or transformed neuronal celllines are amenable to high throughput screening, but do not replicatethe electrophysiological state-dependent signaling of primary neuronalcultures and are unlikely to adequately model the subtle alterations inthis signaling that are caused by oligomers during the earliestmanifestations of the disease state (See e.g. Görtz P, Fleischer W,Rosenbaum C, Otto F, Siebler M. Neuronal network properties of humanteratocarcinoma cell line-derived neurons. Brain Res. 2004 Aug. 20;1018(1):18-25). For this reason, primary neuronal cultures were chosenbecause of their ability to be used in high throughput screens andfidelity to what occurs in vivo.

Reduced formazan was first visible in intracellular vesicles (FIG. 1A).Example of neurons filled with labeled vesicles following endocytosis ofdye and reduction to an insoluble purple product. (Scale bar=20 micronsin FIG. 1A). Eventual formazan exocytosis was accelerated via Abetaoligomers in mature hippocampal neurons in vitro (FIG. 1B). Examplephotomicrograph of neurons covered with insoluble purple dye that havebeen extruded via exocytosis. The dye precipitated in the aqueousenvironment of the culture and formed needle-shaped crystals on thesurface of the neuron. (FIG. 1B). Endocytosis rate was altered in thepresence of Abeta oligomers. (FIG. 1C) Exocytosis rate was altered inthe presence of Abeta oligomers (FIG. 1D).

Since synaptic and memory deficits, and not widespread cell death,predominate at the earliest stages of Alzheimer's disease, assays thatmeasure these changes can be used to discover small molecule inhibitorsof oligomer activity. The MTT assay can be used as a measure of toxicityin cultures. Yellow tetrazolium salts were endocytosed by cells andreduced to insoluble purple formazan in the endosomal pathway. The levelof purple formazan was a reflection of the number of activelymetabolizing cells in culture, and reduction in the amount of formazanwas taken as a measure of cell death or metabolic toxicity in culture.When observed through a microscope, the purple formazan was firstvisible in intracellular vesicles that fill the cell (FIG. 1A). Overtime, the vesicles were exocytosed and the formazan precipitated asneedle-shaped crystals on the outer surface of the plasma membrane asthe insoluble formazan was exposed to the aqueous media environment(FIG. 1B). Cells respond to sublethal levels of Abeta oligomers byselectively accelerating the exocytosis rate of reduced formazan, whileleaving endocytosis rate unaffected, which can be seen in mature primaryneurons in vitro and quantified these morphological shifts via automatedmicroscopy and image processing. At a given point in time followingtetrazolium salt addition to the culture well, vehicle-treated cells hadthe appearance of those in FIG. 1A, while Abeta oligomer-treated cellshad the appearance of those in FIG. 1B. Under these circumstances, therewas no overall change in the total amount of reduced formazan, simply ashift in its morphology. This assay is sensitive to low levels ofoligomers that do not cause cell death.

Evidence suggests that Abeta oligomer-mediated reduction in neuronalsurface receptor expression mediated by membrane trafficking are thebasis for oligomer inhibition of electrophysiological measures ofsynaptic plasticity (LTP) and thus learning and memory (See Kamenetz F,Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S,Malinow R. APP processing and synaptic function. Neuron. 2003 Mar. 27;37(6):925-37; and Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T,Sisodia S, Malinow R. AMPAR removal underlies Abeta-induced synapticdepression and dendritic spine loss. Neuron. 2006 Dec. 7; 52(5):831-43).Measuring membrane trafficking rate changes induced by oligomers viaformazan morphological shifts has been used in cell lines to discoverAbeta oligomer-blocking drugs [Maezawa I, Hong H S, Wu H C, Battina S K,Rana S, Iwamoto T, Radke G A, Pettersson E, Martin G M, Hua D H, Jin LW. A novel tricyclic pyrone compound ameliorates cell death associatedwith intracellular amyloid-beta oligomeric complexes. J Neurochem. 2006July; 98(1):57-67; Liu Y, Schubert D. Cytotoxic amyloid peptides inhibitcellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reduction by enhancing MTT formazan exocytosis. J Neurochem. 1997December; 69 (6):2285-93; Liu Y, Dargusch R, Banh C, Miller C A,Schubert D. Detecting bioactive amyloid beta peptide species inAlzheimer's disease. J Neurochem. 2004 November; 91(3):648-56; Liu Y,Schubert D. Treating Alzheimer's disease by inactivating bioactiveamyloid beta peptide. Curr Alzheimer Res. 2006 April; 3(2):129-35; RanaS, Hong H S, Barrigan L, Jin L W, Hua D H. Syntheses of tricyclicpyrones and pyridinones and protection of Abeta-peptide induced MC65neuronal cell death. Bioorg Med Chem Lett. 2009 Feb. 1; 19(3):670-4.Epub 2008 Dec. 24; and Hong H S, Maezawa I, Budamagunta M, Rana S, ShiA, Vassar R, Liu R, Lam K S, Cheng R H, Hua D H, Voss J C, Jin L W.Candidate anti-Abeta fluorene compounds selected from analogs of amyloidimaging agents. Neurobiol Aging. 2008 Nov. 18. (Epub ahead of print)]that lower Abeta brain levels in rodents in vivo [Hong H S, Rana S,Barrigan L, Shi A, Zhang Y, Zhou F, Jin L W, Hua D H. Inhibition ofAlzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitroand in vivo. J Neurochem. 2009 February; 108(4):1097-1108].

The exocytosis assay was adapted for use with mature primary neuronalcultures grown for 3 weeks in vitro. Abeta oligomers caused adose-dependent decrease in the amount of intracellular vesicles (puncta)filled with reduced purple formazan (Figure. 2A, squares; 304 dosecorresponds to image in FIG. 2C) as measured via image processing usinga Cellomics VTI automated microscopy system. Increasing the amount ofAbeta oligomers eventually resulted in overt toxicity. Thus, theconcentration of neuroactive Abeta oligomers was much lower than thatcausing cell death. This decrease can be blocked by addingstoichiometric amounts of anti-Abeta monoclonal antibody 6E10 (IgG) tothe cultures prior to oligomer addition (FIG. 2A, circle; the circlecorresponds to image in FIG. 2D; antibody alone [down triangle] has noeffect on the neurons). Several compounds were tested that have beenreported to block the effects of Abeta oligomers, including the sugaralcohol scyllo-inositol (AZD-103), the nAChR antagonist hexamethoniumbromide, and the NMDAR antagonists MK-801 and none were active (Feniliet al., '07, Calabrese et al., '06, LeCor et al., '07).

The assay was optimized for performance in 384-well microtiter plateswith automated liquid handling robotics for compound formatting andassay plate stamping, routinely achieving statistically significanttwo-fold separation between vehicle and Abeta oligomer-treated neurons(Student's t-test, unequal variance). Compounds were added to neuronsfirst, then oligomers were added. When configured in this manner theassay was able to detect compounds that act via disruption of oligomers,inhibition of oligomer binding to neurons, or counteraction of signaltransduction mechanisms of action initiated by oligomer binding.

Compounds were considered active if they significantly blockAbeta-mediated changes in membrane trafficking, but do not significantlyaffect membrane trafficking when dosed on their own. An example is shownin FIG. 2B; CT0109 inhibits oligomer effects on membrane traffickingwith an EC50 of 704.

CT0109 is 4-(3-(4-(trifluoromethyl)benzylamino)butyl)-2-methoxyphenol,the structure of which is:

FIG. 2A shows dose-dependent decrease of intracellular formazan-filledvesicles (puncta) caused by Abeta 42 oligomer treatment acceleration ofexocytosis (squares). Oligomer effects were blocked by anti Abeta IgG(circle and up triangle; circle refers to stoich amount of IgG, i.e.,304 of Aβ and 1.5 μM of IgG; up triangle refers to substoich IgG, i.e.,304 of Aβ and 0.5 μM of IgG). IgG itself (down triangle) has no effect.FIG. 2B shows CT0109, which inhibits oligomer effects on membranetrafficking FIG. 2C shows representative micrographs of 21 DIVhippocampal neurons in vitro showing oligomer effects membranetrafficking (corresponding to data point 30 μM in FIG. 2A); and FIG. 2Dshows blockade by anti-Abeta antibodies (corresponding to the circle inFIG. 2A). Data were the average of 3 experiments. Scale bar=20 micron inFIG. 2D.

Example FF Fear Conditioning Assay

CT0109 was tested in an animal model of a memory-dependent behavioraltask known as fear conditioning. The study protocol was designed basedon published protocols (See e.g. Puzzo D, Privitera L, Leznik E, Fa M,Staniszewski A, Palmeri A, Arancio O. Picomolar amyloid-beta positivelymodulates synaptic plasticity and memory in hippocampus. J Neurosci.2008 Dec. 31; 28(53):14537-45). The formation of contextual memories isdependent upon the integrity of medial temporal lobe structures such asthe hippocampus. In this assay mice were trained to remember that aparticular salient context (conditioned stimulus; CS) is associated withan aversive event, in this case a mild foot shock (the unconditionedstimulus, US). Animals that show good learning will express an increasein freezing behavior when placed back into the same context. Thisfreezing is absent in a novel context. Increased freezing in the contextindicates strong hippocampal-dependent memory formation in animals.Memory tested in Fear Conditioning is sensitive to elevations of solubleAβ. FIG. 3 shows the results of administration of Abeta oligomers (barlabeled with “a”) during training results in memory deficits whenanimals are tested 24 later, compared to vehicle administration (barlabeled with “b”). CT0109 was effective at stopping Abeta oligomermediated effects on membrane trafficking (Figure. 3). When administeredto animals prior to Abeta oligomer administration, CT0109 blockedoligomer effects on memory in a dose-dependent manner. The compoundcompletely blocked oligomer-mediated memory deficits at the 2 pmol dose(FIG. 3, bar labeled with “d”). This behavioral efficacy demonstratesthat the membrane trafficking assay is able to predict which compoundswill be efficacious in treating the behavioral memory loss caused byoligomers. The fear condition model for memory was performed asdescribed herein.

FIG. 3 shows that Abeta produces significant deficits in memoryformation vs. vehicle (p<0.05) in the contextual fear conditioningmemory task. FIG. 3 shows that the 2 pmol dose of CT0109+Abeta (200 nM)completely blocked the effect of Abeta on memory (p<0.05, one way ANOVA,post hoc comparison with Bonferroni correction). No effect of compoundalone was observed (data not shown). No adverse behavioral changes wereobserved at any dose.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. Therefore, the spirit and scope of the invention should not belimited to the description of the preferred versions described herein.

All features disclosed in the specification, including the abstract anddrawings, and all the steps in any method or process disclosed, may becombined in any combination, except combinations where at least some ofsuch features and/or steps are mutually exclusive. Each featuredisclosed in the specification, including abstract and drawings, can bereplaced by alternative features serving the same, equivalent or similarpurpose, unless expressly stated otherwise. Thus, unless expresslystated otherwise, each feature disclosed is one example only of ageneric series of equivalent or similar features. Various modificationsof the invention, in addition to those described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Each reference cited in the present application is herein incorporatedby reference in its entirety.

1. A compound of Formula I-0:

or pharmaceutically acceptable salt thereof, wherein:

is a single bond or a double bond; R¹ is H, CH₃, CF₃, F, Cl, Br, or—OCF₃; R² is H, C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl, or C6-10aryl, wherein each of the C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl,or C6-10 aryl is substituted by 0, 1, 2, 3, 4, or 5 substituents eachindependently selected from OH, amino, halo, C1-6 alkyl, C1-6 haloalkyl,C1-6 alkoxy, and C1-6 haloalkoxy; R³ is NR^(3a)R^(3b); R^(3a) is H, C1-6alkyl, C1-6 haloalkyl, cycloalkylalkyl, C3-7 cycloalkyl, arylalkyl, orC6-10 aryl, wherein each of the C1-6 alkyl, C1-6 haloalkyl,cycloalkylalkyl, C3-7 cycloalkyl, or C6-10 aryl is substituted by 0, 1,2, 3, 4, or 5 substituents each independently selected from OH, amino,halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, and C1-6 haloalkoxy;R^(3b) is H, C1-6 alkyl, C1-6 haloalkyl, cycloalkylalkyl,cycloalkylalkyl, C3-7 cycloalkyl, arylalkyl, or C6-10 aryl, wherein eachof the C1-6 alkyl, C1-6 haloalkyl, cycloalkylalkyl, C3-7 cycloalkyl,arylalkyl, or C6-10 aryl is substituted by 0, 1, 2, 3, 4, or 5substituents each independently selected from OH, amino, halo, C1-6alkyl, C1-6 haloalkyl, C1-6 alkoxy, and C1-6 haloalkoxy; or R^(3a) andR^(3b) together with the N atom to which they are attached form a 4-,5-, 6- or 7-membered heterocycloalkyl group that is substituted with 0,1, 2, 3, 4, or 5 substituents each independently selected from OH,amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, andheterocycloalkyl; and R⁴ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇cycloalkyl, or C₆₋₁₀ aryl, wherein each of the C₁₋₆ alkyl, C₁₋₆haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl is substituted by 0, 1, 2, 3,4, or 5 substituents each independently selected from OH, amino, halo,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.
 2. Thecompound of claim 1 of the Formula I:


3. The compound of claim 1 of the Formula Ia-0, Ia, Ib-0, or Ib:

or pharmaceutically acceptable salt thereof.
 4. The compound of claim 1of the Formula Ia-1-0, Ia-1, Ia-2-0, or Ia-2:

or pharmaceutically acceptable salt thereof.
 5. The compound of claim 1of the Formula Ib-1-0, Ib-1, Ib-2-0, or Ib-2:

or pharmaceutically acceptable salt thereof.
 6. The compound of claim 1or pharmaceutically acceptable salt thereof, wherein R¹ is H, CH₃, orCF₃.
 7. The compound of claim 1 or pharmaceutically acceptable saltthereof, wherein R¹ is CH₃ or CF₃.
 8. The compound of claim 1 orpharmaceutically acceptable salt thereof, wherein R¹ is CH₃.
 9. Thecompound of claim 1 or pharmaceutically acceptable salt thereof, whereinR¹ is F, Cl, or Br.
 10. The compound of claims 1-9 or pharmaceuticallyacceptable salt thereof, wherein R² is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl.
 11. The compound of claim 1 orpharmaceutically acceptable salt thereof, wherein R² is H, C₁₋₆ alkyl,or C₃₋₇ cycloalkyl.
 12. The compound of claim 1 or pharmaceuticallyacceptable salt thereof, wherein R² is H or C₁₋₆ alkyl.
 13. The compoundof claim 1 or pharmaceutically acceptable salt thereof, wherein R² is Hor methyl.
 14. The compound of claim 1 or pharmaceutically acceptablesalt thereof, wherein R² is H.
 15. The compound of claim 1 orpharmaceutically acceptable salt thereof, wherein: R^(3a) is H, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl; and R^(3b) is H,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl.
 16. Thecompound of claim 15 or pharmaceutically acceptable salt thereof,wherein R^(3a) is H; and R^(3b) is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, orC₃₋₇ cycloalkyl.
 17. The compound of claim 15 or pharmaceuticallyacceptable salt thereof, wherein R^(3a) is H; and R^(3b) is C₁₋₆ alkylor C₁₋₆ haloalkyl.
 18. The compound of claim 15 or pharmaceuticallyacceptable salt thereof, wherein R^(3a) is H; and R^(3b) is C₁₋₆ alkyl.19. The compound of claim 15 or pharmaceutically acceptable saltthereof, wherein R^(3a) and R^(3b) together with the N atom to whichthey are attached form pyrrolidinyl, piperidinyl, piperazinyl, ormorpholinyl, each substituted with 0, 1, 2, 3, 4, or 5 substituents eachindependently selected from OH, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, aryl, arylalkyl, heteroaryl,heteroarylalkyl, cycloalkyl, and heterocycloalkyl.
 20. The compound ofclaim 15 or pharmaceutically acceptable salt thereof, wherein R^(3a) andR^(3b) together with the N atom to which they are attached formpyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each substitutedwith 0, 1, 2, 3, 4, or 5 substituents each independently selected fromOH, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆haloalkoxy,phenyl, and benzyl.
 21. The compound of claim 1 or pharmaceuticallyacceptable salt thereof, wherein R⁴ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₃₋₇ cycloalkyl, or C₆₋₁₀ aryl.
 22. The compound of claim 1 orpharmaceutically acceptable salt thereof, wherein R⁴ is H, C₁₋₆ alkyl,or C₃₋₇ cycloalkyl.
 23. The compound of claim 1 or pharmaceuticallyacceptable salt thereof, wherein R⁴ is H or C₁₋₆ alkyl.
 24. The compoundof claim 1 or pharmaceutically acceptable salt thereof, wherein R⁴ is Hor methyl.
 25. The compound of claim 1 or pharmaceutically acceptablesalt thereof, wherein R⁴ is H.
 26. A compound selected from:N-isobutyl-2-methyl-6-p-tolylhept-2-en-4-amine; and(6S)—N-isobutyl-2-methyl-6-p-tolylhept-2-en-4-amine; or pharmaceuticallyacceptable salt thereof.
 27. The compound of claim 26 orpharmaceutically acceptable salt thereof, wherein the compound orpharmaceutically acceptable salt thereof has a purity of greater than80%, 90%, 95%, or 99% by weight.
 28. A compound or pharmaceuticallyacceptable salt thereof formed by a process comprising: (a) reacting aturmeric oil with 4-isobutylamine under reactive amination condition toproduce a crude product, wherein the ratio of the ginger oil to4-isobutylamine about 3.4:1 by weight; and (b) isolating a compositioncomprising at least 80%, 85%, 90%, or 95% by weight of a compound or apharmaceutically acceptable salt thereof, wherein: the compound has ananalytical mass spectrum peak of [M+H⁺] at about 274.3.
 29. The compoundof claim 28 or pharmaceutically acceptable salt thereof wherein the ¹HNMR (500 MHz, CDCl₃) spectrum of the compound comprises the peaks atabout the following chemical shifts: δ 7.11, 7.09, 5.23, 3.72, 2.94,2.51, 2.34, 2.31, 1.92, 1.72, 1.71, 1.58, 1.29, 1.27, and 0.92.
 30. Thecompound of claim 28 or pharmaceutically acceptable salt thereof whereinthe ¹³C NMR (125 MHz, CDCl₃) spectrum of the compound comprises peaks atabout the following chemical shifts: δ 144.3, 135.4, 135.0, 129.0,126.9, 126.7, 67.0, 49.3, 46.4, 35.1, 32.5, 25.8, 21.0, 20.7, and 18.4.31. A compound or pharmaceutically acceptable salt thereof formed by aprocess comprising: (a) reacting a turmeric oil with 4-isobutylamineunder reactive amination condition to produce a crude product, whereinthe ratio of the ginger oil to 4-isobutylamine about 3.4:1 by weight;and (b) isolating a composition comprising at least 80%, by weight of acompound or a pharmaceutically acceptable salt thereof, wherein: thecompound has an analytical mass spectrum peak of [M+H⁺] at about 276.3.32. The compound of claim 31 or pharmaceutically acceptable salt thereofwherein the ¹H NMR (500 MHz, CDCl₃) spectrum of the compound comprisesthe peaks at about the following chemical shifts: d 5.77, 5.65, 5.45,5.23, 3.96, 2.94, 2.93, 2.51, 2.31, 2.30, 2.05, 1.92, 1.72, 1.71, 1.58,1.29, 1.27, 0.92.
 33. The compound of claim 31 or pharmaceuticallyacceptable salt thereof wherein the ¹³C NMR (125 MHz, CDCl₃) spectrum ofthe compound comprises peaks at about the following chemical shifts: d144.5, 135.4, 131.0, 127.9, 126.7, 120.8, 66.7, 49.3, 46.4, 35.1, 32.5,25.8, 21.8, 21.79, 20.7, and 18.4. 34-39. (canceled)
 40. Apharmaceutical composition comprising a compound of claim 1 orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 41. A method of inhibiting, treating, and/orabatement of cognitive decline and/or Alzheimer's disease in a patientcomprising administrating to the patient2-methyl-6-p-tolylhept-2-en-4-ol, (6S)-2-methyl-6-p-tolylhept-2-en-4-ol,or a pharmaceutically acceptable salt thereof, or a compound of claim 1or a pharmaceutically acceptable salt thereof.
 42. The method of claim41 wherein the method of inhibiting, treating, and/or abatement ofcognitive decline and/or Alzheimer's disease comprises inhibiting,treating, or abatement of one or more symptoms of cognitive declineselected from the group consisting of memory loss, confusion, impairedjudgment, personality changes, disorientation, and loss of languageskills.
 43. The method of claim 41 wherein the method of inhibiting,treating, and/or abatement of cognitive decline and/or Alzheimer'sdisease comprises one or more of: (i) restoration of long termpotentiation; and/or (ii) inhibiting, treating, and/or abatement ofneurodegeneration; and/or (iii) inhibiting, treating, and/or abatementof general amyloidosis; and/or (iv) inhibiting, treating, and/orabatement of one or more of amyloid production, amyloid assembly,amyloid aggregation, amyloid oligomer binding, and amyloid deposition;and/or (v) inhibiting, treating, and/or abatement of the activity/effectof one or more of Abeta oligomers on a neuron cell.
 44. The method ofclaim 41 wherein the method of inhibiting, treating, and/or abatement ofcognitive decline and/or Alzheimer's disease comprises inhibiting,treating, and/or abatement of one or more of amyloid production, amyloidassembly, the activity/effect of one or more of Abeta oligomers on aneuron cell, amyloid aggregation, amyloid binding, and amyloiddeposition.
 45. The method of claim 41 wherein the method of inhibiting,treating, and/or abatement of cognitive decline and/or Alzheimer'sdisease comprises inhibiting, treating, and/or abatement of one or moreof the activity/effect of one or more of Abeta oligomers on a neuroncell, amyloid aggregation, amyloid binding, and amyloid deposition.46-50. (canceled)